- PA5-26323 - Invitrogen Antibodies GPX1 antibody

Naguib S, Backstrom JR, Gil M, Calkins DJ, Rex TS
Redox biology 2021 Jun;42:101883

Migliaccio V, Scudiero R, Sica R, Lionetti L, Putti R
PloS one 2019;14(4):e0215955

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of HepG2 cells using a GPX1 polyclonal antibody (Product # PA5-26323) at a dilution of 1:10-50, followed by a fluor-conjugated goat anti-rabbit secondary antibody (green). Nuclei were stained with DAPI (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry analysis of GPX1 in HepG2 cells. Samples were incubated in GPX1 polyclonal antibody (Product # PA5-26323) followed by Alexa Fluor 488-conjugated goat anti-rabbit lgG (green). DAPI was used to stain the cell nuclear (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of GPX1 in formalin-fixed and paraffin-embedded human hepatocarcinoma. Samples were incubated with GPX1 polyclonal antibody (Product # PA5-26323) which was peroxidase-conjugated to the secondary antibody, followed by DAB staining. This data demonstrates the use of this antibody for immunohistochemistry; clinical relevance has not been evaluated.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of HeLa cells using a GPX1 polyclonal antibody (Product # PA5-26323) (bottom) compared to a negative control cell (top) at a dilution of 1:10-50, followed by a FITC-conjugated goat anti-rabbit antibody

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry of (overlay histogram) of GPX1 in HepG2 cells (green line). Samples were incubated with GPX1 polyclonal antibody (Product # PA5-26323) using a dilution of 1:25 dilution for 60 min at 37°C followed by Goat-Anti-Rabbit IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed at 1:200 dilution for 40 min at 37°C. The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then incubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the primary antibody. Isotype control antibody (blue line) was rabbit IgG (1 μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry of (overlay histogram) of GPX1 in HepG2 cells (green line). Samples were incubated with GPX1 polyclonal antibody (Product # PA5-26323) using a dilution of 1:25 dilution for 60 min at 37°C followed by Goat-Anti-Rabbit IgG, DyLight® 488 Conjugated Highly Cross-Adsorbed at 1:200 dilution for 40 min at 37°C. The cells were fixed with 2% paraformaldehyde (10 min) and then permeabilized with 90% methanol for 10 min. The cells were then incubated in 2% bovine serum albumin to block non-specific protein-protein interactions followed by the primary antibody. Isotype control antibody (blue line) was rabbit IgG (1 μg/1x10^6 cells) used under the same conditions. Acquisition of >10, 000 events was performed.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 2 ROS and antioxidant proteins increased at 1-wk and 2-wks post-IOP elevation. A) Representative fundus images of DHE fluorescence in all groups. B) Quantification of DHE fluorescence at 1-4 wks post-IOP elevation in comparison to saline-injected controls, showing increases in the microbead injected mice at all time points, **p < 0.001, ****p < 0.0001. C) Fluorescence micrographs of retinas labeled with DAPI (blue) and nitrotyrosine (green). Inset shows double-labeling with anti-nitrotyrosine (green) and anti-NeuN (red). D) Quantification of nitrotyrosine immunofluorescence showing increases at all timepoints after IOP elevation, ***p < 0.001, ****p < 0.0001. E-G) Quantification of fold changes in PCR microarray for: E) GSH-related genes; F) Peroxiredoxin-related genes; and G) SOD-related genes. Dotted line indicates threshold for increase compared to saline. H) Confirmatory qPCR for fold change over saline in antioxidant gene transcription, *p < 0.05, **p < 0.01. I) Representative western blots for beta-actin, PRDX6, SOD3 and GPX1. J-L) Quantification of PRDX6, SOD3 and GPX1, respectively, after normalization to beta-actin, *p < 0.05, **p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Fig. 2

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 PI3K-dependent NRF2 phosphorylation. A) Representative western blots of PI3K and pPI3K B) Quantification of pPI3K to total PI3K, ****p < 0.00001. C) Experiment timeline. D) Representative western blots of pNRF2 to total NRF2. E) Quantification of pNRF2 to total NRF2, ****p < 0.00001. F) Quantification of antioxidant gene transcription shown as fold change over saline, *p < 0.05, ***p < 0.0001. G) Representative western blots for beta-actin, PRDX6, SOD3 and GPX1. H-J) Quantification of PRDX6, GPX1 and SOD3, respectively, after normalization to beta-actin, *p < 0.05, ***p < 0.0001. Fig. 5

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 Decreased antioxidants and increased ROS, axon degeneration, and visual function deficits in ocular hypertensive Nrf2 KO mice. A) Representative fundus images of DHE fluorescence in saline and microbead-injected wildtype and Nrf2 KO mice. B) Quantification of DHE fluorescence at 2-wks post-IOP elevation in both strains of mice. C) Quantification of antioxidant gene transcription shown as fold change over respective saline groups, *p < 0.05, **p < 0.001, ***p < 0.0001. D) Representative western blots for beta-actin, PRDX6, SOD3 and GPX1. E-G) Quantification of PRDX6, GPX1 and SOD3, respectively, after normalization to beta-actin, **p < 0.001. H) Representative micrographs of wildtype and Nrf2 KO optic nerves at 2-wks post-IOP elevation. Scale bar applies to all micrographs. Arrows indicate degenerative axons. I) Quantification of total axons in the optic nerves, showing a decrease only in ocular hypertensive Nrf2 KO mice, **p < 0.001. J) Quantification of number of degenerative axons, showing an increase in ocular hypertensive Nrf2 KO mice, ***p < 0.0001. K) Quantification of the N1 amplitude of the VEP. L) Quantification of the PhNR amplitude. Note: all wild-type data shown in this Figure was copied from Fig. 2 for purposes of comparison. Fig. 6

PA5-26323